1Department of Dermatology, Indiana University School of Medicine, Indianapolis, IN, USA.

Abstract

Ultraviolet B radiation (UVB) is a potent stimulator of epidermal cytokine production which has been implicated in photoaggravated dermatoses. In addition to cytokines such as tumor necrosis factor-alpha (TNF-alpha), UVB generates bioactive lipids including platelet-activating factor (PAF). Our previous studies have demonstrated that UVB-mediated production of keratinocyte TNF-alpha is in part due to PAF. The current studies use a human PAF-receptor (PAF-R) negative epithelial cell line transduced with PAF-Rs and PAF-R-deficient mice to demonstrate that activation of the epidermal PAF-R along with UVB irradiation results in a synergistic production of TNF-alpha. It should be noted that PAF-R effects are mimicked by the protein kinase C (PKC) agonist phorbol myristic acetate, and are inhibited by pharmacological antagonists of the PKC gamma isoenzyme. These studies suggest that concomitant PAF-R activation and UVB irradiation results in a synergistic production of the cytokine TNF-alpha which is mediated in part via PKC. These studies provide a novel potential mechanism for photosensitivity responses.

Effect of various stimuli along with UVB on TNF-α protein production in KB cells. PAF–R-expressing KBP and control PAF–R-negative KBM cells were treated with 100 nm of the metabolically stable PAF-R agonist CPAF, 500 nm of the phorbol ester PMA, 500 nm of the calcium ionophore A23187, 600 J m−2 UVB, ethanol vehicle control (CON) or the combination of UVB 30 min after either vehicle, CPAF, ionophore or PMA. At 6 h after treatment, supernatants were collected and levels of TNF-α protein were measured by ELISA. The data represent mean ± SD TNF-α protein of duplicate samples from a representative experiment from four separate experiments.

Effect of various stimuli along with UVB on TNF-α mRNA production in KB cells. PAF–R-expressing KBP and control PAF–R-negative KBM cells were treated with 100 nm of the metabolically stable PAF-R agonist CPAF, 500 nm of the phorbol ester PMA, 600 J m−2 UVB, ethanol vehicle control (CON) or the combination of UVB 30 min following either CPAF or PMA. At 4 h after treatment, mRNA was isolated and qRT-PCR for TNF-α and 18S performed. The data represent mean amount of TNF-α mRNA relative to 18S ± SD of duplicate samples from a representative experiment from three separate experiments.

Time course of CPAF along with UVB on TNF-α mRNA production in KBP cells. PAF–R-expressing KBP cells were treated with ethanol vehicle or 100 nm of the metabolically stable PAF-R agonist CPAF followed by sham or 600 J m−2 UVB 30 min later. At various times, mRNA was isolated and qRT-PCR for TNF-α and 18S mRNA performed. The data represent mean amount of TNF-α mRNA relative to 18S ± SD of duplicate samples from a representative experiment from two separate experiments.

Effect of CPAF along with UVB on TNF-α mRNA production in murine epidermis. The dorsal ears of wild-type and PAF-R−/− mice were treated with either intradermal injection of 50 μL of either CPAF or 0.5% ethanol control in PBS. In some experiments PAF-R−/− mice were treated with topical application of 10 μg of PMA in 10 μL or ethanol vehicle on the dorsal ears. Fifteen minutes following the treatments, the ears were treated with 1200 J m−2 UVB. Four hours later mRNA was extracted from the auricular epidermis, and homogenized and total RNA was isolated. RNA was reverse transcribed and qRT-PCR performed. The expression levels of TNF-α mRNA were normalized to the expression levels of 18S. The data listed are the mean ± SEM normalized TNF-α mRNA from duplicate samples from n = 6–8 mice. *P < 0.05; **P < 0.01; ***P < 0.001 in comparison with vehicle-treated skin.